PROJECT SUMMARY RESEARCH PROJECT 2 Respiratory viruses, such as respiratory syncytial virus (RSV), are major triggers of asthma exacerbation in children and adults. The overarching hypothesis of this project is that airway epithelial cells (AECs) coordinate responses to respiratory virus infection and aeroallergens. Moreover, intrinsic differences between AECs of asthmatic and healthy children change both innate and adaptive responses during infection and exposure to allergens. We hypothesize that infection of AECs from asthmatic children with RSV or HRV, in combination with allergen exposure, induces resident innate cells, including dendritic cells (DCs) and innate lymphoid cells (ILCs), to trigger type-2 responses. We will test this idea using AECs isolated from asthmatic and healthy children, grown at air/liquid interface (ALI) and infected with respiratory viruses in the context of allergen challenge. We will examine the response of DCs and ILCs co-cultured with these AECs, and their role in promoting type-2 inflammation through the following aims: Aim 1. Test hypothesis that AECs control local DC maturation and function. Since AECs in asthmatics may be ?leaky? due to barrier defects, we will first determine whether more antigen is transferred by asthmatic than healthy AECs to DCs using modified RSV strains. Then, we will test whether AECs from asthmatic children differ in promoting DC differentiation and function, and how virus infection and allergen exposure impact this interaction. Finally, we will assess the ability of DCs exposed to AECs from asthmatic children to drive CD4 T cell differentiation and proliferation in co-culture assays. Aim 2. Test hypothesis that after allergen exposure and viral infections, asthmatic and healthy AECs differentially impact lung ILC2 through lipid mediators and vasoactive intestinal peptide. We will assess the role of AECs in regulating ILC2s, which are the most abundant ILCs in the lung. We expect to find that AECs from asthmatic children enhance ILC2 function during viral infection and allergen challenge. In our preliminary data, we found two distinct subsets of human lung ILC2s that differentially express receptors for oxysterols, retinoic acid, and vasoactive intestinal peptide. Thus, we propose to define the impact of these lipidic and neuropeptide mediators on the activation of different ILC2 populations. Aim 3. Test hypothesis that ICOS+ ILCs regulate lung immune responses. We made the novel observation that the lung contains an unusual subset of ICOS+ ILCs. These cells do not fit into the ILC1, ILC2, ILC3 paradigm, express a marker of regulatory lymphocytes and are enriched in an immunosuppressive environment associated. Thus, we hypothesize that these cells may have regulatory functions. To test this, we propose to isolate lung ICOS+ ILCs and define their transcriptome profile ex vivo as well as their functional capabilities in vitro, including cytokine and chemokine secretion. Moreover, we propose to test their responsiveness to stimuli derived from ALI cultures exposed to HDM and/or viruses.